Molecular monitoring by CDKN2A/p16INK4A and RB1 gene methylation in breast cancer

SUMMARY OBJECTIVE: This prospective study aimed to provide a comprehensive analysis of the methylation status of two pivotal genes, CDKN2A/p16 INK4A (cyclin-dependent kinase inhibitor 2A) and RB1 (retinoblastoma transcriptional corepressor 1), in breast cancer patients. METHODS: Samples were obtained from 15 women diagnosed with breast cancer and who underwent a total mastectomy. DNA was extracted from the tumor, non-tumor tissue, and peripheral blood (circulating cell-free DNA). The methylation pattern of cell-free DNA extracted from blood collected on the day of mastectomy was compared with the methylation pattern of cell-free DNA from blood collected 1 year post-surgery. The methylation analysis was carried out by sodium bisulfite conversion and polymerase chain reaction, followed by electrophoresis. RESULTS: Methylation of CDKN2A/p16 INK4A was identified in 13 tumor samples and 12 non-tumor tissue samples. Two patients exhibited CDKN2A/p16 INK4A methylation in the cell-free DNA of the first blood collection, while another showed methylation only in the cell-free DNA of the subsequent blood collection. Regarding RB1, 11 tumors and 8 non-tumor tissue samples presented methylation of the gene. CONCLUSION: This study presents a novel approach for monitoring breast cancer patients through the analysis of cell-free DNA methylation. This analysis can detect changes in methylation patterns before any visible sign of cancer appears in breast tissue and could help predict the recurrence of malignant breast tumors.


INTRODUCTION
Activation of oncogenes and inactivation of tumor suppressor genes are genetic oscillations related to cancer development 1,2 .DNA methylation, which is an epigenetic mechanism, can influence the expression of tumor suppressors and genes related to uncontrolled cell proliferation.The mechanism can inactivate gene expression and consequently prevent protein synthesis.
The CDKN2A/p16 INK4A (cyclin-dependent kinase inhibitor 2A), located at chromosome 9 (9p21.3), is a tumor suppressor gene that encodes p16 INK4A , a protein involved in cell cycle regulation 1,3,4 .The p16 INK4A participates in the G1-to-S-phase checkpoint and may interrupt the cell cycle in response to various stressors, leading to the inhibition of cell proliferation 1,5 .Additionally, p16INK4A promotes apoptosis of tumor cells and can increase sensitivity to chemotherapy in breast cancer 3 .
An investigation found a positive correlation between hypermethylation of CDKN2A/p16 INK4A and breast cancer progression and also verified that CDKN2A/p16 INK4A hypermethylation impacts the tumor's grade and stage 3 .
The RB1 (retinoblastoma transcriptional corepressor 1) is also a tumor suppressor gene and is located on chromosome 13 (13q14.2).RB1 encodes the retinoblastoma protein (Rb), and alterations in its tumor suppressor pathway can be considered a potential risk for the development of breast cancer [6][7][8] .RB1 can be inactivated by several mechanisms, including alterations in phosphorylation, viral oncoproteins, and promoter hypermethylation 7 .
The analysis of methylation may be performed in circulating cell-free DNA (cfDNA) using liquid biopsy, a promising method for early detection and monitoring of breast cancer 9 .Liquid biopsy is non-invasive and easy to perform, and analysis

Data collection and ethical aspects
The recruitment occurred between October 2018 and July 2021.The medical records served as the basis for obtaining clinical and demographic data.The institutional ethics committee approved the study protocol (certificate: CAAE nº 91406118.6.0000.5257from August 29, 2018).

Material collection
After pre-mastectomy, samples of the tumor, non-tumor tissue, and 5 mL of peripheral blood were collected immediately for DNA analysis.Approximately 1 year after the surgery, patients were invited for a new blood collection, and a second cfDNA analysis was performed.

Extraction of DNA from the tumor, surrounding tissue, and blood serum
The DNA extraction from tumor and non-tumor tissues was performed by the phenol:chloroform method 13 , using the Ultra Pure™ Phenol:Chloroform:Isoamyl Alcohol, from Invitrogen™, Cat.No. 15593-031.Quick-gDNA™ MiniPrep Kit (Zymo Research) Cat.No. D3024 was used for cfDNA extraction from blood serum according to the manufacturer's protocol.

Methylation mechanism
Sodium bisulfite conversion and MSP (methylation-specific polymerase chain reaction (PCR)) were adopted to analyze DNA methylation using the EZ DNA Methylation-Gold TM Kit, Cat.No: D5005, Zymo Research, according to the manufacturer's protocol.

Polymerase chain reaction
The DNA integrity was confirmed through amplification of exon 5 of the p53 gene as previously described 14 .For the CDKN2/p16 INK4A amplification, two pairs of primers were used as follows: CDKN2A/p16 INK4A -U (unmethylated) forward, 5′-TATTAGAGGGTGGGGTGGATTGT-3′ and CDKN2A/ p16 INK4A -U reverse, 5′-CAACCCCAAACCACAACCATAA-3′ p r o d u c i n g a f r a g m e n t o f 1 5 1 b a s e p a i r s a n d C D K N 2 A / p 1 6 I N K 4 A -M ( m e t h y l a t e d ) f o r w a r d , 5′-TTATTAGAGGGTGGGGCGGATCGC-3′ and CDKN2A/ p16 INK4A -M reverse, 5′-ACCCCGAACCGCGACCGTAA-3′ producing a fragment of 150 base pairs 15 .The polymerase used for the MSP was the GoTaq G2 Hot Start Green Master Mix, Cat.No: M7422, Promega.PCR conditions were as follows: initial denaturation at 96°C for 7 min, followed by 35 cycles of 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min.The final extension was performed at 72°C for 7 min.RB1-U (unmethylated) forward, 5′-GGGAGTTTTGTGGATGTGAT-3′ and RB1-U reverse, 5′-ACATCAAAACACACCCCA-3′ producing a fragment of 172 base pairs and RB1-M (methylated) forward, 5′-GGGAGTTTCGCGGACGTGAC-3′ and RB1-M reverse, 5′-ACGTCGAAACACGCCCCG-3′ producing a fragment of 172 base pairs 16 .The polymerase used for the MSP was the GoTaq G2 Hot Start Green Master Mix, Cat.No: M7422, Promega.PCR conditions were as follows: initial denaturation at 96°C for 7 min, followed by 35 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min.The final extension was performed at 72°C for 5 min.

Gel electrophoresis and staining
After amplification, PCR products were separated on polyacrylamide gels at a concentration of 10%.Each electrophoretic run had the addition of a negative control and a DNA marker.Gels were stained by the silver nitrate method, allowing visualization of the DNA bands as previously described 17 .In short, DNA fixation with methanol and acetic acid occurred in the first step, followed by impregnation with silver nitrate in the next step and, finally, with sodium hydroxide (NaOH) and formaldehyde to reveal the DNA bands.
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RESULTS
According to the data obtained from medical records, all patients had advanced disease (stage III or IV). Figure 1 shows the methylation statuses of CDKN2A/p16 INK4A and RB1 in the tumor, non-tumor tissue, and cfDNA (from the first and second blood collections).CDKN2A/p16 INK4A methylation was detected in 13 tumors and 12 non-tumor tissue samples.Only two patients (2 and 3) presented CDKN2A/p16 INK4A methylation in the cfDNA from the blood of the first collection, and only patient 14 presented it in the cfDNA from the blood of the second collection.Furthermore, RB1 methylation was detected in 11 tumors and 8 non-tumor tissues.Patient 8 showed a weak band of RB1 methylation in the cfDNA from the blood of the first collection and a strong band of methylation in the cfDNA from the blood of the second collection.Moreover, patient 14 showed a weak band of methylation only in the cfDNA from the second collection.

DISCUSSION
This study investigated the methylation statuses of CDKN2A/ p16 INK4A and RB1 genes in breast cancer patients.DNA from the tumor, non-tumor tissue, and blood serum (cfDNA) was analyzed.The analyses showed that the vast majority of tumor samples had methylation of both CDKN2A/p16 INK4A (13/15) and RB1 (11/15).This result was expected as methylation of these genes is related to breast cancer development, as pointed out by Cheng et al. 3 and Yao 7 .Regarding the non-tumor tissue, the majority of samples (although slightly less than in tumors) also showed methylation of CDKN2A/p16 INK4A (12/15) and RB1 (8/15).This confirms that non-tumor tissue, although apparently free of malignancy, is part of the tumor microenvironment.These changes were not detected by histopathological examination, but  the molecular analysis of methylation indicated that the tissue considered tumor-free was already in the process of molecular modification with potential for future cancerization.
Only a small number of cfDNA samples showed methylation of CDKN2A/p16 INK4A (2 and 3 in the first collection, and none in the second collection) and RB1 (8 in the first collection and 8 and 14 in the second collection).This must be related to the chemotherapy treatment given to patients before mastectomy.As highlighted by Kujala et al. 11 , chemotherapy reduces the concentration of tumor DNA in the blood.
Patient 14 had no methylation in either CDKN2A/p16 INK4A or RB1 in the cfDNA of the first blood collection.In contrast, both genes were methylated in the cfDNA of the second blood collection.In addition, patient 8 showed a weak band of RB1 methylation in the cfDNA from the blood of the first collection and a strong band in the cfDNA of the second collection.The fact that the bands corresponding to methylation are stronger in the cfDNA from the second blood collection suggests a possible recurrence of the disease.Consequently, an increased concentration of tumoral DNA released into the bloodstream is observed, which is visualized as stronger DNA bands by gel electrophoresis.

CONCLUSION
This study presented a novel approach for monitoring breast cancer patients through the assessment of methylation in cfDNA.Once liquid biopsy is non-invasive and easy to perform, the long-term follow-up of patients is facilitated.The cfDNA analysis proposed here can detect changes in methylation patterns before any visible sign of disease appears in breast tissue.This suggests that the study of cancer-related gene methylation in cfDNA could help predict the recurrence of malignant breast tumors.

Figure 1 .
Figure 1.Methylation of CDKN2A/p16 INK4A and RB1 genes in breast cancer patients.CDKN2A/p16 INK4A : cyclin-dependent kinase inhibitor 2A; RB1: retinoblastoma transcriptional corepressor 1; T: tumor; NT: non-tumor tissue; cfDNA I: circulating cell-free DNA of the first blood collection; cfDNA II: circulating cell-free DNA of the second blood collection; M: methylated DNA; U: unmethylated DNA; C-: negative control; †: death; Ø: no return to the second blood collection for other reason than death.Numbers correspond to patients.

Table 1 .
Methylation panel of CDKN2A/p16 INK4A gene in the tumor, non-tumor tissue, and cell-free DNA of breast cancer patients.